Gene Center Munich and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany.
Gene Center Munich and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany.
Cell Rep. 2020 Sep 29;32(13):108190. doi: 10.1016/j.celrep.2020.108190.
Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1, MIS12, and NDC80 complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association.
着丝粒上的动粒是介导有丝分裂过程中染色体正确分离的着丝点的大型蛋白组装体。外动粒 KNL1、MIS12 和 NDC80 复合物组装 KMN 网络，该网络包含微管结合和纺锤体组装检查点信号的位点。将微管拉力传递到芽殖酵母点着丝粒的 KMN 网络的构建知之甚少。在这里，我们通过对从酿酒酵母中分离出的 KMN 复合物进行质谱分析，确定了 225 个蛋白间交联，描绘了外动粒组装的 KMN 亚基连接。Nsl1 和 Mtw1 的 C 末端基序通过 Kre28 招募 SPC105 复合物，这两个基序都有助于以前报道的 Dsn1 C 末端通过 Dsn1 C 末端稳定 NDC80 复合物。我们表明，三个 C 末端 MTW1 亚基基序的中心介导了 KMN 网络的协同稳定，这一稳定作用通过 NDC80-SPC105 直接关联得到增强。
